An ideal anti-cancer drug would be one that is active only when inside cancer cells and that targets essential components or disrupts essential processes of those cells. Mitochondria, supplying much of the cellular energy and key regulators of apoptosis, are emerging as effective targets that may provide the cell selectivity desired for anti-cancer therapy (Don and Hogg, 2004, Armstrong, 2006).
Cytotoxic drugs that act by selectively affecting mitochondria in cancer cells, ‘mitocans’, are proving to be highly attractive for the treatment of cancer since these compounds can work as potent anti-cancer agents with little or no side effects, as reported in animal studies (Ko et al, 2004). Mitocans disrupt the energy producing systems of cancer cell mitochondria, leading to increased reactive oxygen species (ROS) and activation of the mitochondrial dependent cell death signaling pathways inside cancer cells.
Mitocans include drugs affecting the following mitochondrial associated activities: hexokinase inhibitors; electron transport/respiratory chain blockers; activators of the mitochondrial membrane permeability transition pore targeting constituent protein subunits, either the voltage dependent anion-selective channel (VDAC) or adenine nucleotide transporter (ANT); inhibitors of Bcl-2 anti-apoptotic family proteins and Bax/Bid pro-apoptotic mimetics. Two prime examples of mitocans with little or no side effects are 3-bromopyruvate (3-BP) and α-tocopheryl succinate (α-TOS), both of which induce apoptosis in cancer cells with much greater efficacy than in normal cells (Ko et al, 2004, Geschwind et al, 2002, Xu et al, 2005, Neuzil et al, 2001, 2004).
One group of mitocans includes pro-oxidant analogues of vitamin E (Wang et al 2006). The great promise of pro-oxidant vitamin E analogues, epitomized by α-TOS, as anti-cancer drugs stems from studies with experimentally contrived cancers, such as human xenografts growing in nude mice, where they have been shown to suppress malignancy (reviewed in Neuzil et al, 2004). Such studies include colorectal (Neuzil et al, 2001, Weber et al, 2002) and lung carcinomas (Quin et al, 2005), melanomas (Malafa et al, 2002), as well as mesotheliomas (Tomasetti et al, 2004, Stapelberg et al, 2005). α-TOS has also been shown to promote breast cancer dormancy (Malafa et al, 2000) and suppress colon cancer metastases into the liver (Barnett et al, 2002).
Although vitamin E (α-tocopherol, α-TOH) acts as a potent anti-oxidant in cells, α-TOS, an esterified, redox-silent and pro-oxidant analogue of vitamin E, has distinctive properties. In contrast to α-TOH, α-TOS acts as a strong cell stressor, causing rapid production of ROS in a range of different cancer cell lines (Neuzil et al, 2004, Weber et al, 2003, Wang et al, 2005, Swettenham et al, 2005, Stapelberg et al, 2005). α-TOS also has the ability to bind to and inhibit Bcl-2/Bcl-xL (Done et al, 2006). Evidence to date suggests that the cancer cell-specific nature of α-TOS and the lack of toxic effect on normal cells occurs because normal cells are endowed with greater anti-oxidant defenses (Allen and Balin, 2003, Safford et al, 1994, Church et al, 1993) and/or contain high levels of esterases that inactivate α-TOS by releasing the succinate moiety, thereby producing the redox-active, non-apoptogenic α-TOH (Fariss et al, 2001, Neuzil et al, 2004, Neuzil and Massa, 2005).
Naturally occurring vitamin E consists of a mixture of eight compounds which differ by the methylation patterns of the chromanol ring (α-, β-, γ-, δ-tocopherol) and the number of double bonds of the phytyl side-chain (α-, β-, γ-, δ-tocotrienol). The role of these molecules as lipophilic anti-oxidants in vitro and in vivo is widely accepted. In addition, the non-anti-oxidant properties of members of the VE family have also been investigated (Azzi et al, 2002).
The vitamin E molecule can be divided into three different domains. The Functional Domain (1) arises from the substitution pattern at position C6 of the chromanol ring. This position determines whether the molecule behaves as redox-active or redox-silent, since a free hydroxyl group is essential for vitamin E to function as an anti-oxidant. The well documented anti-oxidant properties of the four tocopherol isomers resulted in their application in cancer clinical trials. None of these studies showed a positive outcome concerning the use of free tocopherols in cancer prevention (Pham and Plakogiannis, 2005). However, certain chemical modifications at C6 led to ethers (RO—), esters (RCOO—) and amides (RCONH—) that proved to be potent anti-neoplastic agents. See Table 1 below.
TABLE 1Anti-proliferative activity of vitamin E analogues.Compounds are sorted by the Signaling Domain.FunctionalSignallingHydrophobicIC50CellNrDomain I (R1)Domain IIDomain III (R2)[μM]typeRef1−O2CCH2CH2COO—43Jurkat, HBT11, MCF7, MCF7- C3Birringer et al, 20032CH3COO—a3−O2CCH═CHCOO—224−O2CCH2CH(CH3)COO—b5−O2CCH2(CH2)2COO—b6−O2CCH2CH(CH3)CH2COO—b7−O2CCH2C(CH3)2CH2COO—b8−O2CC(CH3)2CH2CH2COO—b9H3COOCCH2CH2COO—b10−O2CCOO—cB16-F1/Kogure11−O2CCH2COO—nudeet a.,mice200512−O2CCH2CH2CONH—13Jurkat,Tomic-13−O2CCH═CHCONH—2U937,Vatic et14H3COOCCH2CH2CONH—>100Meso-2al, 200515+NH3—CH2COO—aMCF7Arya et16+NH3Lys(NH3)COO—12al, 199517Lys-Lys(Lys)COO—a18CH3O—aJurkatNeuzilet al,2001b19CH3CH2COO—dA549Yano etal, 200520−O2CCH2CH2CH2O—eLNCaP,Wu etPC-3al,MDA-2004;MB-453Nishikawa et al,200321−O2CCH2O—fMDA-Shun etMB-435,al, 2004MCF722−O2CCH2—15-20gMCF7Shiau etal, 200623(PET)O2CCH2CH2COO—hlungYouk etcarcinoal, 2005ma cells/nudemice24−O2C(CH2)5COO—aC1271Kogure25C2H5OOCCH2CH2COO—aet al,26nicotinic acida200427−O2CCH2CH(SePh)COO—?prostateVraka etal, 200628all-trans retinoic acid0.1-1NB4,Makishi299-cis retinoic acidbHT93ma etal,1996,199830HOPO2O—bRASMC,Muntea31Toc-OPO2O—bTHP-1nu et al,2004 32−O2CCH2CH2COO—50% of α-TOSJurkat, HVT11, MCF7, MCF7- C3, U937, Meso-2Birringer et al, 2003; Tomic- Vatic et al, 2005 33−O2CCH2CH2COO—bJurkat, HBT11, MCF7, MCF7- C3Birringer et al, 2003; Vraka et al, 2006 34−O2CCH2CH(SePh)COO—bprostateVraka etal, 2006 35−O2CCH2CH2COO—66Jurkat, HBT11, MCF7, MCF7- C3Birringer et al, 2003; Tomic- Vatic et al, 2005 36−O2CCH═CHCOO—49Jurkat,Tomic-37−O2CCH2CH2CONH—20U937,Vatic et38−O2CCH═CHCONH—9Meso-2al, 200539H3COOCCH2CH2COO—aBirringeret al,200340HO—bPC-3Galli etal, 2004aNo effect;binhibition of cell proliferation;cmuch more cytotoxic than α-TOS;dless effective than 54;ethe ether analogue is less effective than a-TOS itself;fcomparable to α-TOS;gEC50 [μg/ml];hmore efficient than α-TOS.
TABLE 2Anti-proliferative activity of vitamin E analogues with a modified HydrophobicDomain.FunctionalSignallingHydrophobicIC50Nr.Domain I (R1)Domain IIDomain III (R2)[μM]Cell typeRef41−O2CCH2CH2COO—COO—Jurkat, HBT11, MCF7, MCF7-C3Birringer et al, 2003 42HO—aLNCaP, PC-3Shiau et al, 2006 43−O2CCH2CH2COO—4-944−O2CCH2CH2O—4-8 45−O2CCH2CH2COO—8-19 46CH3>10047+NH3Lys(NH3)COO—CH2—OH194MCF7Arya et48CH2—O-nC5H1122al, 199549CH2—OC(O)nC4H91550CH2—O-cholic acid451HO—CH2CH2COO−bPC-3Galli etal, 2004 52HO—CH2CH2COO−caNo effect;bweak inhibition at 50 μM;c82% inhibition at 10 μM.
TABLE 3Anti-proliferative activity of vitamin E analogues. Compounds are sorted by theSignaling Domain.FunctionalSignallingHydrophobicIC50Nr.Domain I (R1)Domain IIDomain III (R2)[μM]Cell typeRef.53HO—210MDA-MB- 435Guthrie et al, 199714MCF7110B16(F10)He et al, 1997 54CH3CH2COO—aA549Yano et al, 2005 55HO—bJurkat, HBT11, MCF7, MCF7-C3Birringer et al, 2003 56HO—4neoplastic + SA mammary epithelial cellsShah and Sylvester, 2005 15cMCF7He et al,1997dJurkat,BirringerHBt11,et al,MCF7,2003MCF7-C320B16(F10)He et al,199757−O2CCH2CH2COO—eJurkat,BirringerHBT11,et al,MCF7,2003MCF7-C3fprostateVraka etal, 200658−O2CCH2CH(SePh)COO—fprostateVraka etal, 2006 59HO—10B16(F10)He et al, 1997bMDA-MB-Shun et435,al, 2004MCF715cMCF7Nesaretnam etal, 1996 60HO—0.9B16(F10)He et al, 1997aCytotoxic in 0-40 μM range;bvery potent;ccomplete inhibition;dcomparable to α-TOS;e2-fold more potent than γ- tocotrienol;finhibition of cell proliferation.
The second domain, termed the Signaling Domain (II), exhibits some activities that are independent of the anti-oxidant properties of the tocopherols. These properties derive from the methylation pattern of the aromatic ring. For example, α-tocopherol has been reported to inhibit protein kinase C (PKC) by decreasing diacylglycerol (DAG) levels, while other tocopherols with similar anti-oxidant capabilities (e.g., β-tocopherol) do not inhibit PKC. Thus, the PKC inhibitory activity of α-tocopherol is independent of its anti-oxidant capacity (Tasinato et al, 1995; Kunisaki et al, 1995). In some cases, however, the biological activity of the various tocopherols is influenced by structural differences in the Signaling Domain, which do indeed have a profound impact on their anti-oxidant activity against certain species. γ-Tocopherol, for example, is a much better scavenger of reactive nitrogen oxide species (e.g., peroxynitrite) than α-tocopherol. Hence, the γ-molecule, which lacks a methyl group at C5, is readily nitrated at that site (Morton et al, 2002; Christen et al, 1997).
The lipophilic side chain of vitamin E isomers distinguishes between tocopherols with saturated isoprenyl units and tocotrienols with unsaturated isoprenyl units. The Hydrophobic Domain (III) determines whether the molecule can bind to lipoproteins and membranes respectively, or be degraded by phase 1 enzymes (Birringer et al, 2002; Neuzil and Massa, 2005).
Many tocopherol derivatives with a modified hydroxyl group have been tested for their pro-apoptotic activity (Table 1). The most prominent derivative tested has been α-TOS (entry 1) bearing a succinylester at position C6 of the chromanol ring. Due to its low pKa (<6), α-TOS is fully deprotonated under physiological conditions, leading to a detergent-like molecule which destabilizes mitochondrial membranes and has an effect on complex II. Dicarboxylic esters of tocopherols present the best studied compounds for structure-activity relationship (SAR). Strong apoptogens included α-tocopherol succinate (1), oxalate (10), and malonate (11), the latter two inducing non-selective cytotoxicity in mice inoculated with B16-F1 melanoma cells (Kogure et al, 2005). Even greater pro-apoptotic activity has been observed for unsaturated dicarboxylic acids like α-tocopheryl maleate (3) (Birringer et al., 2003) and α-tocopheryl fumarate. Increasing the chain length of the dicarboxylic acid led to decreased activity as shown for glutaric acid (5), methylated glutaric acids (6, 7, 8) (Birringer et al, 2003) with the pimelic acid (24) (Kogure et al, 2004) exhibiting no activity at all.
It has been established that the whole α-TOS molecule is necessary for its full apoptosis inducing activity (Birringer et al, 2003). Esterification of the free carboxyl group leads to non-charged derivatives without pro-apoptotic activity (9, 25). Aliphatic carboxylic acid esters, such as tocopheryl acetate and propionate (19), respectively, were inactive as was the methyl ether (18). Oral administration of α-TOS is not effective since the compound is cleaved by intestinal esterases (Wu et al, 2004b; Cheeseman et al, 1995). To overcome the problem of ester bond cleavage, compounds (20, 21) and a side chain-truncated derivative (42) have been synthesized, replacing the ester bond with an ether bond, since the latter is resistant to hydrolysis (Wu et al, 2004b; Nishikawa et al, 2003; Shun et al, 2004; Shiau et al, 2006). It should be noted that the replacement of the ether bond by a methylene group is sufficient to accelerate apoptosis (22) (Sanders et al, 2001).
When the ester bond is replaced by an amide bond, further enhancement of pro-apoptotic activity was observed (12, 13, 37, 38) (Tomic-Vatic et al, 2005). Again the unsaturated amides (13, 38) were superior to the saturated amides. The rationale for introducing an amide bond in place of the ester was based on the well-established fact that anilinic amides are much less prone to hydrolysis than the corresponding phenolic esters. Enhancing the stability of these tocopheryl ester derivatives would protect these molecules in viva, allowing them to stay intact longer, thereby increasing their bioavailability. The isosteric replacement of the esters by amides makes that linkage less prone to enzymatic hydrolysis as well. Several nonspecific esterases exist in the intestinal mucosal cells and in the blood. In contrast, peptidases exhibit a much narrower specificity. For example, prodrugs with an amino acid in an amide linkage are more stable in the intestine and blood than their corresponding ester analogues (Sugawara et at 2000).
The last group of compounds consisted of a series of lysine α-tocopheryl esters with a positively charged N-terminus (15-17). The hydrophilic ammonium functionality exerted similar pro-apoptotic effects to its carboxylate counterpart, suggesting a general motif is required for activity that consists of a lipophilic side chain and a hydrophilic head group. However, succinyl esters of long chain aliphatic alcohols (e.g., phytol and oleol) did not show any activity (Birringer et al, 2003).
A general SAR can be drawn from the data shown in Table 1:    1. To gain profound pro-oxidant and pro-apoptotic activity, modifications of the Functional Domain I required a hydrophilic head group consisting of a dissociated acid or a charged ammonium group.    2. The chain length and the degree of unsaturation of the Functional Domain determined the apoptogenic activity. Conformational restrictions appeared to potentiate the activity.    3. The chemical linkage of the Functional Domain is not limited to esters, and other functionalities prevented enzymatic degradation of the derivatives.
The substitution pattern of the chromanol ring is often not merely related to the anti-oxidant properties of the tocopherols (Azzi et al, 2002). Different biochemical observations emphasize the role of α-tocopherol in signaling and metabolic processes. Thus, α-tocopherol is selectively recognized in the liver by α-tocopherol transfer protein (α-TTP), a 32 kDa protein with a high affinity for α-tocopherol relative to the other tocopherols and tocotrienols. The relative affinities for α-TTP decrease with the loss of methylation of the chromanol ring (α-tocopherol 100%, β-tocopherol 38%, γ-tocopherol 9% and δ-tocopherol 2%) (Hosomi et al, 1997). The recently discovered tocopherol associated proteins (TAPs) show similar preferences in tocopherol binding (Yamauchi et al, 2001). In endothelial cells, thrombin-induced PKC activation and endothelin secretion are inhibited by α-tocopherol but not by β-tocopherol (Martin-Nizard et al, 1998). At the transcriptional level α-tocopherol causes up-regulation of α-tropomyosin expression (Aratri et al, 1999) and down regulation of LDL scavenger receptors SR-A and CD36, whereas β-tocopherol is ineffective (Ricciarelli et al, 2000; Devaraj et al, 2001). In addition, the substitution pattern is likely responsible for the rate of side chain degradation because in cell culture, γ- and δ-tocopherol are degraded much faster than α- or β-tocopherol (Birringer et al, 2001). Succinylation of the four tocopherol isomers produces the compounds 1, 32, 33 and 35. It is not surprising that of these, α-TOS (1) possesses the highest apoptogenic activity tested, followed by β-TOS (32), γ-TOS (33) and δ-TOS (35) as the least effective (Birringer et al, 2001). In general, the more highly methylated members of the tocopherol family are the most potent, but this trend is reversed for the tocotrienols (see below).
Succinylation of Trolox, a water soluble vitamin E derivative with a shortened side chain, resulted in the complete loss of pro-apoptotic activity. SAR experiments of various tocopherol succinates bearing truncated phytol side chains (Table 2, 43, 44, 45) revealed the highest level of apoptogenic activity in prostate cancer cells was obtained with derivatives where the side chain length was two isoprenyl units (43, 44). Computer assisted molecular modeling and co-immunoprecipitation experiments showed that the binding of Bak BH3 peptide to Bcl-xL and Bcl-2 was inhibited by the tocopherol analogues (Shiau et al, 2006). Central requirements for anti-neoplastic activity were succinylation of the chromanol ring and a minimum chain length of one isoprenyl unit (42, 46). A series of tocopheryl lysine esters with ether/ester linked Domain III side chains also showed a negative correlation between chain length and IC50 (47-50) (Arya et al., 1998).
Tocotrienols are efficient anti-cancer agents and their pro-apoptotic property may be related to the inactivation of the Ras family of proteins. Tocotrienols exhibit their pro-apoptotic activity without modifications of the Functional Domain. The hierarchy in the Signaling Domain is also reversed, making δ-tocotrienol (59) the most potent agent in the murine B16-F10 melanoma cell model, followed by γ- (56) and α-tocotrienol (53) (Table 3; He et al, 1997). Interestingly, desmethyl tocotrienol (60), lacking all aromatic methyl groups, shows even higher activity with an IC50 of 0.9 μM. This compound has been isolated from rice bran (Qureshi et al, 2000). A direct inhibitory action of tocotrienols has been proposed because the membrane anchoring cysteine residue of Ras proteins is modified by a common structural element, a farnesyl chain. Thus, Ras farnesylation and RhoA prenylation was inhibited by tocotrienols in A549 cells, a human lung adenocarcinoma cell line containing an activating ms mutation (Yano et al, 2005). To expand the short in vivo half life of tocotrienols, functional domains have been introduced. These modifications also enhanced the antiproliferative activity of the molecules (54, 57, 58). Truncation of the side chain also improved activity, similar to that found for compound 55.
A number of compounds where modifications have been made to the Functional Domain exhibit anti-proliferative activity and provide additional specialized properties. For example, α-Tocopheryl polyethylene glycol succinate (23) has been used as a vehicle for drug delivery systems. This compound was shown to possess anti-cancer activity against human lung carcinoma cells implanted in nude mice. The apoptosis inducing efficacy of the compound was not due to its increased uptake into cells, but rather due to an increased ability to generate reactive oxygen species (Youk et al, 2005). α-Tocopheryl phosphate (30) is believed to result from metabolism occurring during tocopherol-associated signaling (Negis et al, 2005). Mixtures of 30 and di-α-tocopheryl phosphate (31) inhibited proliferation in rat aortic smooth muscle cells and in human THP-1 monocytic leukaemia cells (Munteanu et al, 2004). The authors proposed that tocopheryl succinate and tocopheryl maleate may act in cancer cells by mimicking and substituting for tocopheryl phosphate and thereby cause the permanent activation of cellular signals.
Two experimental α-tocopheryl esters of all-trans retinoic acid (28) and 9-cis retinoic acid (29), respectively, have been used to reduce proliferation of acute promyelocytic leukaemia cells (Makishima et al, 1998). Trans-activation experiments with retinoid receptor responsive reporter constructs revealed that both of these compounds acted as agonists for retinoic acid receptors (RARs), γ-Carboxyethyl hydroxychroman (52), a degradation product of γ-tocopherol often found secreted in the urine, is able to reduce cell proliferation of PC-3 prostate cancer cells by inhibiting cyclin D1 expression (Galli et al, 2004).